The present invention relates to novel ultra high affinity neutralizing antibodies.
The current incidence of infection caused by resistant or difficult to control microbes has created a need for newer approaches to controlling such organisms, as well as to treating those already infected.
Among the more difficult infectious agents to control and treat are the viruses. For example, respiratory syncytial virus (RSV) is the major cause of acute respiratory illness in young children admitted to hospitals and the major cause of lower respiratory tract infection in young children. A major obstacle to producing an effective vaccine against such agents as RSV has been the issue of safety. Conversely, the use of immunoglobulins against such viral agents has proven of some value. For example, studies have shown that high-titred RSV immunoglobulin was effective both in prophylaxis and therapy for RSV infections in animal models.
An alternative approach has been the development of antibodies, especially neutralizing monoclonal antibodies, with high specific neutralizing activity. One drawback to this route has been the need to produce human antibodies rather than those of mouse or rat and thus minimize the development of human anti-mouse or anti-rat antibody responses, which potentially results in further immune pathology.
An alternative approach has been the production of human-murine chimeric antibodies in which the genes encoding the mouse heavy and light chain variable regions have been coupled to the genes for human heavy and light chain constant regions to produce chimeric, or hybrid, antibodies.
In some cases, mouse CDRs have been grafted onto human constant and framework regions with some of the mouse framework amino acids being substituted for correspondingly positioned human amino acids to provide a xe2x80x9chumanizedxe2x80x9d antibody. [Queen, U.S. Pat. Nos. 5,693,761 and 5,693,762]. However, such antibodies contain intact mouse CDR regions and have met with mixed effectiveness, producing affinities often no higher than 107 to 108 Mxe2x88x921 .
A humanized anti-RSV antibody with good affinity has been prepared and is currently being marketed. [See: Johnson, U.S. Pat. No. 5,824,307]
The production of ultra high affinity antibodies would be desirable from the point of view of both the neutralizing ability of such an antibody as well as from the more practical aspects of needing to produce less antibody in order to achieve a desirable degree of clinical effectiveness, thereby cutting costs of production and/or allowing a greater degree of clinical effectiveness for administration in the same volume.
The present invention relates to high affinity neutralizing antibodies and active fragments thereof exhibiting affinity constants of at least 1010 Mxe2x88x921, and even 1011 Mxe2x88x921, and more specifically to such a neutralizing monoclonal immunoglobulin, including antibodies and/or active fragments thereof, wherein the antibody and/or fragment has human constant regions.
The present invention solves the above-mentioned problems by providing high affinity neutralizing antibodies without the presence of intact mouse CDR regions that cause human anti-mouse antibody reactions (HAMA) and with sufficiently high affinity neutralizing activity to reduce cost and efficacy of overall production.
One aspect of the present invention relates to high affinity neutralizing antibodies with affinity constants of at least 1010 Mxe2x88x921, and even 1011 Mxe2x88x921, and with specificity towards specific antigenic determinants, such as those exhibited by virus-expressed proteins.
One object of the present invention is to provide such high affinity neutralizing antibodies with specificity toward antigens produced by viruses, such as where such antigens are expressed by virus-infected cells in a mammal, especially a human.
In one such embodiment, the high affinity neutralizing immunoglobulin, including antibodies and/or active fragments thereof, of the present invention, and active fragments thereof, are specific for respiratory syncytial virus (RSV), most especially for the F antigen expressed by said RSV and also expressed on the surfaces of cells infected with RSV (the presence of which antigen on the cell surface causes fusion of the cells into syncytia).
Thus, in one embodiment, a high affinity neutralizing immunoglobulin, including antibodies and/or active fragments thereof, of the present invention binds to the same epitope on RSV as the antibody whose light chain variable chain has the sequence of SEQ ID NO: 1 (shown in FIG. 1A) and whose heavy chain variable chain has the sequence of SEQ ID NO: 2 (shown in FIG. 1B).
It is an object of the present invention to provide ultra high affinity neutralizing antibodies having substantially the framework regions of the immunoglobulin disclosed in FIG. 1 (with the same specificity of that immunoglobulin, which is an anti-RSV antibody structure) but wherein the immunoglobulins, including antibodies and active fragments thereof, of the present invention contain one or more CDRs (complementarity determining regions) whose amino acid sequences are independent of those in the so-called reference antibody, although said sequences may, in some cases, differ by no more than one amino acid and this may be limited to a difference in only one of said CDR regions.
In a preferred embodiment of the present invention, the novel immunoglobulins of the present invention will differ from the antibody of FIG. 1 (hereafter, the xe2x80x9cbasic antibodyxe2x80x9d or xe2x80x9creference antibodyxe2x80x9d or xe2x80x9creference immunoglobulinxe2x80x9d) only in the sequences of one or more of the CDRs and, in a most preferred embodiment these differences occur only in CDRs L2, L3, H1, and H3.
Especially preferred embodiments of the present invention have the framework sequences depicted in FIG. 1, thus having the heavy and light chain variable sequences depicted in FIGS. 3, 4, 5, 6, and 7.
In one embodiment, the high affinity neutralizing antibodies of the invention include a human constant region.
In a preferred embodiment, a high affinity RSV-neutralizing antibody of the invention, including active fragments thereof, with an affinity constant (Ka) of at least as high as 1010 Mxe2x88x921, and even 1011 Mxe2x88x921, is a recombinant immunoglobulin, such as an antibody or active fragment thereof, that includes a human constant region and framework regions for the heavy and light chains wherein at least a portion of the framework is derived from a human antibody (or from a consensus sequence of a human antibody framework), an example of said framework regions depicted for the antibody sequences of FIG. 1.
In one embodiment, all of the framework is derived from a human antibody (or a human consensus sequence).
In another highly specific embodiment, a high affinity RSV-neutralizing antibody, with an affinity of at least 1010 Mxe2x88x921, is a recombinant antibody having a human constant region, one or more CDRs that are derived from a non-human antibody in which at least one of the amino acids in at least one of the CDRs is changed and in which all or a portion of the framework is derived from a human antibody (or a consensus sequence of a human antibody framework).
In a separate embodiment, a humanized neutralizing immunoglobulin that binds to the same epitope as the basic or reference antibody or immunoglobulin whose variable chains are shown in FIG. 1, and that has an affinity of at least 1011 Mxe2x88x921, includes at least one of the following amino acids at the following positions of the CDRs: an alanine at position 2 of CDR H1, a phenylalanine at position 6 of CDR H3, a phenylalanine at position 3 of CDR L2, and a phenylalanine at position 5 of CDR L3. Other embodiments comprise other single amino acid substitutions at these locations.
It is another object of the present invention to provide compositions comprising the immunoglobulins disclosed herein wherein said structures are suspended in a pharmacologically acceptable diluent or excipient.
It is a still further object of the present invention to provide methods of preventing and/or treating respiratory syncytial virus comprising the administering to a patient at risk thereof, or afflicted therewith, of a therapeutically effective amount of a composition containing an immunoglobulin of the invention, such as where said antibodies or active fragments thereof exhibit the specificity and affinity properties disclosed herein for the immunoglobulins of the invention.
The term xe2x80x9cantigenxe2x80x9d refers to a structure, often a polypeptide or protein, present on the surface of a microorganism, such as a virus, for which an antibody has affinity and specificity.
The term xe2x80x9cantigenic determinantxe2x80x9d refers to a specific binding site on an antigenic structure for which an immunoglobulin, such as an antibody, has specificity and affinity. Thus, a particle, such as a virus, may represent an antigen but may have on its surface a number of separate, and different, antigenic sites such as where the virus has a number of different surface proteins and each represents a distinct potential binding site for an immunoglobulin.
The term xe2x80x9cimmunoglobulinxe2x80x9d refers to a protein or polypeptide having specificity and affinity for an antigen or antigenic determinant. This term includes antibodies, commonly depicted as tetrameric, as well as active fragments thereof, such fragments having specificity and affinity for antigens or antigenic determinants. Thus, xe2x80x9cimmunoglobulinxe2x80x9d as used herein includes antibodies and all active fragments thereof.
The term xe2x80x9cantibodyxe2x80x9d refers to a protein or polypeptide having affinity for an antigenic determinant, usually one found on the surface of a microorganism, especially a virus. Such an antibody is commonly composed of 4 chains and is thus tetrameric.
The term xe2x80x9cneutralizing immunoglobulinxe2x80x9d or xe2x80x9cneutralizing antibodyxe2x80x9d refers to the ability of the immunoglobulins, including antibodies, of the present invention to reduce the replication of microorganisms, especially viruses, in organisms as well as in cell cultures. An indication of such ability is the data from the microneutralization assays disclosed hereinbelow. Such a structure usually has both variable and constant regions whereby the variable regions are mostly responsible for determining the specificity of the antibody and will comprise complementarity determining regions (CDRs).
The term xe2x80x9ccomplementarity determining regionxe2x80x9d or xe2x80x9cCDRxe2x80x9d refers to variable regions of either H (heavy) or L (light) chains contains the amino acid sequences capable of specifically binding to antigenic targets. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure. Such regions are also referred to as xe2x80x9chypervariable regions.xe2x80x9d
The term xe2x80x9cactive fragmentxe2x80x9d refers to a portion of an antibody that by itself has high affinity for an antigenic determinant and contains one or more CDRs accounting for such specificity. Non-limiting examples include Fab, F(ab)xe2x80x22, heavy-light chain dimers, and single chain structures, such as a complete light chain or complete heavy chain.
The term xe2x80x9cspecificityxe2x80x9d refers to the ability of an antibody to bind preferentially to one antigenic site versus a different antigenic site and does not necessarily imply high affinity.
The term xe2x80x9caffinityxe2x80x9d refers to the degree to which an antibody binds to an antigen so as to shift the equilibrium of antigen and antibody toward the presence of a complex formed by their binding. Thus, where an antigen and antibody are combined in relatively equal concentration, an antibody of high affinity will bind to the available antigen so as to shift the equilibrium toward high concentration of the resulting complex.
The term xe2x80x9caffinity constantxe2x80x9d refers to an association constant used to measure the affinity of an antibody for an antigen. The higher the affinity constant the greater the affinity of the immunoglobulin for the antigen or antigenic determinant and vice versa. An affinity constant is a binding constant in units of reciprocal molarity. Such a constant is readily calculated from the rate constants for the association-dissociation reactions as measured by standard kinetic methodology for antibody reactions.